OPEN Enriched Environment Inhibits Mouse SUBJECT AREAS: Pancreatic Cancer Growth and PANCREATIC CANCER LIFESTYLE MODIFICATION Down-regulates the Expression of Received Mitochondria-related Genes in Cancer 8 September 2014 Accepted Cells 17 December 2014 Guohua Li1*, Yu Gan1*, Yingchao Fan1, Yufeng Wu1, Hechun Lin1, Yanfang Song1, Xiaojin Cai1, Published Xiang Yu2, Weihong Pan3, Ming Yao1, Jianren Gu1 & Hong Tu1 19 January 2015 1State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University 2 Correspondence and School of Medicine, 25/Ln. 2200 Xietu Road, Shanghai 200032, China, Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, requests for materials 3 China, Blood-Brain Barrier Group, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808, United should be addressed to States. H.T. (tuhong@shsci. org) Psycho-social stress has been suggested to influence the development of cancer, but it remains poorly defined with regard to pancreatic cancer, a lethal malignancy with few effective treatment modalities. In this * These authors study, we sought to investigate the impacts of enriched environment (EE) housing, a rodent model of contributed equally to ‘‘eustress’’, on the growth of mouse pancreatic cancer, and to explore the potential underlying mechanisms through gene expression profiling. The EE mice showed significantly reduced tumor weights in both this work subcutaneous (53%) and orthotopic (41%) models, while each single component of EE (inanimate stimulation, social stimulation or physical exercise) was not profound enough to achieve comparative anti-tumor effects as EE. The integrative transcriptomic and proteomic analysis revealed that in response to EE, a total of 129 genes in the tumors showed differential expression at both the mRNA and protein levels. The differentially expressed genes were mostly localized to the mitochondria and enriched in the citrate cycle and oxidative phosphorylation pathways. Interestingly, nearly all of the mitochondria-related genes were down-regulated by EE. Our data have provided experimental evidence in favor of the application of positive stress or of benign environmental stimulation in pancreatic cancer therapy. ancreatic cancer is one of the most deadly neoplastic diseases, with a 5-year survival rate of below 5%1. Pancreatic cancer is typically asymptomatic in its early stages and is profoundly resistant to conventional P chemo- and radio-therapies. In addition, none of the novel approaches attempted over the past decades for the treatment of pancreatic cancer have been demonstrated to be clinically beneficial or to improve patient survival benefit2. Thus, there is an urgent need to identify new strategies to treat patients with this deadly disease. Recently, there has been increasing interest in the effects of environmental factors, and specifically the physical living and social stimulation, on the development of peripheral cancer. Adverse psycho-social factors, including striking life events, high levels of depressive symptoms, and low levels of social support, have been related to higher cancer incidences, such as those of breast and colon cancer3–5. Psycho-social distress, which is a negative stress associated with exposure to severe aversive and hostile environments6, has also been related to poor survival in cancer patients3,7. Recently, a tumor-promoting effect of the distress response has been demonstrated in a pancreatic cancer xenograft mouse model8, indicating that psycho-social factors may be sufficiently profound to regulate the growth of pancreatic cancer. The housing of laboratory rodents in an enriched environment (EE) is a classical and widely used model for studying environmental impacts in neuroscience. Compared to standard housing conditions, EEs consist of more complex housing with increased space and enhanced social interactions and physical activity9. The EE, which promotes ‘‘eustress’’ or positive psycho-social stress10, has been demonstrated to influence brain structure and SCIENTIFIC REPORTS | 5 : 7856 | DOI: 10.1038/srep07856 1 www.nature.com/scientificreports Figure 1 | Enriched environment confers beneficial effects to brain and behavior in mice. (A) Enriched environment (EE) and standard environment (SE) housing conditions. (B) Schematic diagram of the experimental protocol. Mice were tested for anxiety-like behaviors with the elevated plus-maze (EPM) after 3 weeks (immediately before tumor implantation; C and D) or after 5 weeks (2 weeks after Panc02 tumor implantation; D) following housing in EE or SE. (C) Representative travel pathways of an SE and an EE mouse on the EPM. (D) Comparison of time spent in open arm (top) and distance traveled in open arm (bottom) between the SE and EE mice. More time or increased distance in open arm represents lower levels of anxiety-like behaviors. (E) Real-time analysis of brain BDNF mRNA levels in Panc02 tumor-bearing mice. BDNF mRNA expression significantly increased in the hippocampus (top) and hypothalamus (bottom) of the EE mice. (D, E) Data are presented as the mean 6 SEM; n 5 12 per group. * P , 0.05, ** P , 0.01 versus SE mice. function11. It elicits a number of beneficial effects on the central housing conditions of EE and standard environment (SE), which are nervous system (CNS), such as reduced anxiety levels12,13, enhanced described in detail in the Methods section. The experimental protocol is learning and memory14, induction of hippocampal neurogenesis and schematically diagrammed in Figure 1B. Three-week-old C57BL/6 neural plasticity15,16, and improved recovery from brain injury and mice were randomly assigned to either EE or SE housing for 3 cerebral disorders9,17,18. Interestingly, EE has been associated with weeks. Both EE and SE mice were then given subcutaneous anti-tumor phenotypes and to significantly inhibit tumor growth injections of Panc02 pancreatic cancer cells and returned to their in syngeneic melanoma, colon cancer10, and breast cancer models19. respective homes for 5 additional weeks. Because one of the most These intriguing results provide an experimental indication of the important benefits of EE to the CNS is to reduce the anxiety level of importance of eustress for tumor growth control20. Studies focusing residents12,13, we examined its effects on anxiety-like behaviors of mice on EEs and pancreatic cancer may provide insights to facilitate the by elevated plus maze (EPM), which is a behavioral test commonly improvement of pancreatic cancer interventions. used to assess anxiety levels in rodents. There were no differences in the In the current study, we tested whether EE could influence the behavioral parameters between the SE and EE groups upon study growth of pancreatic cancer in mouse subcutaneous and orthotopical entry. However, mice housed in EE for 3 weeks (immediately before xenograft models. Because EE is a complex stimulation composed of tumor implantation) showed significantly lower levels of anxiety-like several environmental components, we also investigated the ele- behaviors, reflected by longer cumulative time spent in the open arms ments that may play roles in the regulation of cancer growth. (SE 5 53.7 6 5.2 s, EE 5 102.8 6 5.2 s, P , 0.01) and higher Additionally, by comparing the global gene expression profiles of percentages of distances travelled in the open arms (SE 5 8.1 6 xenograft tumors from EE- and non EE-raised mice, we were able 1.0%, EE 5 15.3 6 1.3%, P , 0.01) (Figure 1C & D). The EPMs to demonstrate that mitochondrial metabolic genes were generally were repeated 2 weeks after tumor implantation (following a total of down-regulated by the EE. 5 weeks of EE housing). Consistently, the tumor-bearing mice living in the EE conditions showed decreased trend of anxiety-like behaviors Results with borderline significance (time in open arm: SE 5 50.1 6 10.8 s, EE Establishment of the EE condition for rearing normal and 5 79.2 6 10.3 s, P 5 0.06; percentage of distance travelled in open pancreatic cancer-implanted C57BL/6 Mice. Figure 1A shows the arm: SE 5 7.9 6 1.3%, EE 5 12.1 6 1.7%, P 5 0.07) (Figure 1D). SCIENTIFIC REPORTS | 5 : 7856 | DOI: 10.1038/srep07856 2 www.nature.com/scientificreports Figure 2 | Enriched environment reduced tumor growth in syngeneic mouse models of pancreatic cancer. Mice were housed in the enriched environment (EE) or the standard environment (SE) for 3 weeks and then subcutaneously injected with Panc02 cells (6 3 105 cells per mouse). (A) Growth curve based on the tumor volumes monitored once a week after implantation. (B) Subcutaneous Panc02 tumors dissected at the time of sacrifice. (C) Comparison of tumor weights between the SE and EE mice. EE housing significantly inhibited the growth of the subcutaneous Panc02 tumors. Reduced tumor sizes in EE were associated with the decreased proliferating cell nuclear antigen (PCNA) staining of the Panc02 tumors. (D) Representative immunohistochemical staining of PCNA in the Panc02 tumors from the SE and EE mice. Scale bars: 50 mm. (E) Quantitative analysis of PCNA1 tumor cells. EE- and SE-housed mice were also orthotopically injected with Panc02 cells (1 3 105 cells per mouse). (F) Orthotopic Panc02 tumors dissected at the time of sacrifice. (G) Comparison of orthotopic tumor weights between the SE and EE mice. There were significantly reductions in the tumor weights in the EE-housed mice. (A, C, E, G) Data are presented as the mean 6 SEM; n 5 12 per group. * P , 0.05, ** P , 0.01 versus SE mice. The expression of the brain-derived neurotrophic factor (BDNF) To further confirm the inhibitory effects of EE on the growth of is considered to be an effector immediate-early gene responsive to pancreatic cancer, we established an orthotopic pancreatic cancer EE10. To further confirm the CNS effects of EE in tumor-bearing model in EE- and SE-housed mice.